It is known that some of the hormones circulating in the body fluids show two forms: one is bound to carrier proteins and the other is unbound. The two fractions are in an equilibrium state between themselves and the free fraction is, in many cases, directly responsible of the biological effects. In particular, thyroid hormones, thyroxine (T4) and triiodothyronine (T3) circulating in the blood are mainly bound to carrier proteins by a noncovalent binding. Carrier proteins include an .alpha.-globulin (thyroxine-binding globulin, TBG), binding about 75% of the hormones, a pre-albumin fraction (TBPA) and an albumin fraction (TBA) binding about 15% and 10% of the hormones, respectively. The association constants of T3 and T4 with binding proteins are different: T3-TBG association constant is lower than T4-TBG association constant, T3-TBPA association constant is negligible. Even if very small fractions of T3 and T4 circulate unbound in plasma (FT3 and FT4), only unbound fractions are thought to be biologically active. Evidence has been given that bound hormone fractions cannot pass through cell membranes.
It is therefore essential to correlate the hormone functions with its free fraction rather than with its total amount present in the serum. Variations of the free fractions observed in hypo- and hyperthyroid subjects, are more closely correlated to clinical symptoms than those found for total thyroid hormones. Free fractions levels of hypo- and hyperthyroid subjects do not overleap values of euthyroid subjects, as observed for total hormones level. Therefore, free fraction measurements avoid doubtful diagnostic evaluations occuring in "border-line" zones.
The determination of free T3 and T4 discloses new interesting studies in non thyroidal diseases. For example, an increase of free T4 level is observed in patients affected by angina pectoris. In addition, as free thyroid hormones levels, and particularly free T3level, play an important role in maintaining the homeostasis of hypothalamo-pituitary-thyroid system, a more extensive evaluation of this role in physiological and pathological conditions can be performed.
Interaction between thyroid hormones and binding proteins can be described by a reversible reaction, to which the mass action law can be applied: the equilibrium between the unbound (free) and the bound hormone fraction depends on the association constant of each binding protein and on the number of binding sites. Circulating hormone-protein complexes can be considered as reservoir system to avoid sharp variations of thyroid secretory activity. About 0.01 to 0.04 percent of the total amount of T4 and 0.2 to 0.4 percent of the total amount of T3 are unbound. With regard to hormones capable of binding to serum proteins, for each species of binding site the situation is represented by the following equilibrium ##EQU1## wherein H is the concentration of the free hormone, P.sub.i is the concentration of the free sites of type i, HP.sub.i is the concentration of the bound sites of type i and K.sub.i is the relative association constant.
In this case the concentration of the free hormone is ##EQU2## wherein M.sub.i is the total concentration of bound and non-bound sites of type i. Since the binding sites of a single protein may be of different type as well as the proteins present in the whole system, in a more general way, in the case of n species of binding sites in the serum, the situation at the equilibrium conditions is represented by the following relationship ##EQU3## wherein H is the concentration of the circulating free hormone, M.sub.i and HP.sub.i are as described above and the summations refer to the species of binding sites i in the serum and to the relative association constants toward the hormone.
TBG and TBPA have a single binding site per molecule while albumine has two binding sites with different association constant.
The concentration of a free hormone in a biological fluid is usually determined by multiplying its free fraction percent by the amount of total hormone present in the fluid. Said percentage may be determined, for instance, through dialysis of the serum mixed with a known amount of labelled free hormone. This method applied to thyroxine (T4) is, for instance, described by S. H. Ingbar et al. in J. Clin. Invest. 44, 1679, (1965). However, it does not yield very reliable results since there are several factors which may affect the result such as the presence of I.sup.125 ions which negatively influences the calculation of the fraction of bound hormone to non-bound hormone.
The direct method provided by S. Ellis and R. Ekins (Acta Endocrinologica Suppl. 177, 106, 1973-IX Acta Endocrinologica Congress, Oslo June, 17-21, 1973) for the determination of thyroxine and triiodothyronine requires a good technical experience which is generally not common among the technicians who carry out these kinds of analysis as the method is rather complicated. Also this method is based on dialysis procedures.
In the specific case of T4, two further approaches have been studied. The first one(free T4 index)is based on the determination of the total T4 serum level (according for instance to U.S. Pat. No. 3,659,104) and on the T3 uptake (according, for instance, to U.S. Pat. No. 3,710,117). The second one is based on the complete dissociation of T4 in the presence of labelled T4, followed by absorption on a resin and elution of labelled T4 from the resin by means of a portion of the serum under examination (U.S. Pat. No. 3,941,564).
By using the first approach, the arithmetic product of the T4 total serum level and the T3 uptake is an indirect extimate of free T4. (free T4 index). This extimate is better correlated to the thyroid functionality; however two separated tests are necessary with a magnification of the errors. Both methods, yield an indirect value of free T4. With these methods no information can be obtained about free T3.
For the reasons given above it is evident that only a direct measurement of free T3 and T4 has greater possibility to be utilized for diagnostics purposes.